Identification and analysis of a class 2 alpha-mannosidase from Aspergillus nidulans.
A Class 2 alpha-mannosidase gene was cloned and sequenced from the filamentous fungus Aspergillus nidulans. A portion of the gene was amplified using degenerate oligonucleotide primers which were designed based on similarity between the Saccharomyces cerevisiae vacuolar and rat ER/cytosolic Class 2 protein sequences. The PCR amplification product was used to isolate the full length gene, and DNA sequencing revealed a 3383 bp coding region containing three introns. The predicted 1049 amino acid reading frame contained six potential N-glycosylation sites and encoded a protein of 118 kDa. The protein sequence did not appear to encode a typical fungal signal sequence or membrane spanning domain. Although the cellular location of the A.nidulans mannosidase was not determined, experimental evidence suggested that it was located within a subcellular organelle. The Matchbox sequence similarity matrix indicated that the A.nidulans protein sequence was more highly similar to the rat ER/cytosolic (Rij = 0.33) and S.cerevisiae vacuolar alpha-mannosidases (Rij = 0.43) than the rat and yeast sequences were to each other (Rij = 0.29). These three enzymes were found to be distantly related to other Class 2 sequences, and compose a third subgroup of Class 2 alpha-mannosidases, as shown by ClustalW sequence alignment.
Eades
C J
CJ
Department of Biology, University of Victoria, P.O. Box 3020, Victoria, British Columbia V8W 3N5, Canada.
Gilbert
A M
AM
Goodman
C D
CD
Hintz
W E
WE
D000595:Amino Acid Sequence; D000818:Animals; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015870:Gene Expression; D018390:Gene Targeting; D005800:Genes, Fungal; D008361:Mannosidases / Q000145:classification* / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D043323:alpha-Mannosidase
D017931:DNA Primers; D004271:DNA, Fungal; D008361:Mannosidases; D043323:alpha-Mannosidase